Melanoma develops as melanocytes accumulate genetic and epigenetic abnormalities, typically causing oncogene activation and senescence, followed by escape from senescence and loss of tumor suppression. Members of the Class I MAGE gene family are normally expressed only in developing germ cells and placenta but their expression is activated in many malignancies including melanoma. Class I MAGE protein expression is associated with poor clinical outcomes and resistance to treatment, and knockdown of MAGE proteins increases melanoma cell apoptosis in vitro and decreases growth of MAGE (+) human melanoma cells in immunocompromised mice, suggesting that MAGE proteins play important functional roles in melanoma biology. MAGE proteins bind to and regulate KAP1, a scaffolding protein and ubiquitin E3-ligase that causes localized chromatin compaction and represses gene expression when recruited to specific DNA sites by KRAB-zinc finger transcription factors (KZFs), the largest group of transcription factors in vertebrates. MAGE proteins modify KZF-KAP1 binding to and repression of specific genes, and MAGE expression affects Id1, AP2 and p16, all known to be involved in melanocytic transformation. However, the specific roles of MAGE proteins in the biology of melanoma, their mechanisms of action, and ways to exploit them for treatment have not been not fully characterized. Our overall goal is to understand the role of Class I MAGE expression in melanoma development. We will test the hypothesis that MAGE proteins facilitate melanocyte transformation by regulating expression of genes that cooperate to promote escape from senescence, and increase melanocyte proliferation and tissue invasion. Aim 1: To determine how MAGE affects melanocyte growth and tissue invasion. Aim 1a will test whether MAGE promotes escape from BRAFV600E induced senescence by suppressing p16 via Id1, using selective knockdown of endogenous Id1, p16 and MAGE in BRAFV600E (+) cells, or by ectopic expression of BRAFV600E , Id1, p16, and MAGE, followed by measurement of cell growth and senescence markers. Epigenetic effects of MAGE will be determined by ChIP for MAGE and histone 3 trimethylated on leucine 9 (H3me3K9), a molecular signature of KAP1 induced gene repression. Aim 1b will use a similar strategy to test whether MAGE regulation of AP2 promotes migration and invasion of melanocytes in Boyden chamber and in vitro wound assays. Aim 2: To establish in vivo associations of MAGE expression with melanocyte transformation. This Aim will use state of the art multispectral immunohistology with a unique tissue microarray to test the hypothesis that MAGE is expressed early in melanocyte transformation and that MAGE proteins co-localize with markers of proliferation (Ki67) and are inversely correlated with expression of senescence markers. Aim 3: To test the hypothesis that MAGE expression promotes melanocyte transformation in vivo. Aim 3a will determine whether MAGE affects melanocyte migration or tissue invasion in utero using immunohistology and unique transgenic mice that express melanocyte specific, doxycycline inducible MAGE. Aim 3b will test the hypothesis that MAGE promotes melanocyte transformation by crossing melanocyte MAGE (+) mice into BRAFV600E or NRASQ61K based murine models of oncogene induced melanocytic nevi. Aim 3c will determine the effects of MAGE-A3 expression in early melanoma by crossing MAGE transgenics into melanoma models expressing BRAFV600E or NRASQ61K and showing loss of tumor suppressors PTEN or p16. Outcomes include the rate of transformation, number, and histologic characteristics of resulting melanomas. Assessment of MAGE repression of individual genes will use ChIP, reverse transcription real time quantitative PCR (RT-qPCR) and protein immunoblotting. These studies will benefit veterans by identifying novel targets for treatment and prevention of melanoma.